【摘要】 目的:建立人少突胶质细胞糖蛋白细胞外Ig样结构域(MOGIgd)的硫氧还蛋白(TrxA)融合表达系统,探讨表达产物MOGIgd-TrxA融合蛋白的免疫原性。方法:(1)应用RT-PCR方法,以人脑组织的总RNA为模板扩增出编码MOGIgd的cDNA序列,将MOGIgd cDNA克隆到TrxA融合表达载体pET32a(+)中,重组质粒pET32a-MOGIgd经表型筛选、酶切鉴定及测序鉴定后,转化至BL21(DEB)trxB-宿主菌中经异丙硫代钠-β-D-半乳糖苷(IPTG)诱导表达,并通过金属鳌合亲和层析柱进行纯化。(2)C57BL/6小鼠分为MOGIgd-TrxA组(MOG组)、硫氧还蛋白组(TrxA组)及正常对照组(NC组),12只/组,各组以相应抗原乳剂免疫小鼠制作实验性自身免疫性脑脊髓炎(EAE)模型后观察其临床神经功能和组织病理学改变(HE染色和髓鞘Luxol fast blue染色)评价模型质量。结果:(1)成功获取高产量32 kD MOGIgd-TrxA融合蛋白,以包涵体形式表达,纯化后纯度达95%左右。(2)MOG组小鼠75%(9/12)发病,于免疫后第(12.14±2.04)d发病,呈慢性非缓解型病程;发病动物组织切片HE染色和髓鞘染色显示不同程度炎性细胞浸润和髓鞘脱失。结论:成功建立人MOGIgd的TrxA融合表达系统,表达产物MOGIgd-TrxA融合蛋白可作为免疫原诱导EAE动物模型。更多还原

【Abstract】 AIM:To construct a thioredoxin(TrxA)fusion expression vector expressing the extracellular immunoglobulin domain of myelin oligodendrocyte glycoprotein(MOGIgd)and investigate the immunogenicity of MOGIgd-TrxA fusion protein.METHODS: The cDNA encoding MOGIgd was amplified from human brain tissue by reverse transcription polymerase chain reaction.The cloned MOGIgd cDNA was ligated into the thioredoxin fusion expression vector pET32a(+).Recombinant plasmid was transfected into E.coli BL21(DE3) trxB-,and was induced with isopropyl-β-D-thiogalacto-pyranoside.The MOGIgd-TrxA fusion protein and TrxA protein were purified by metal chelate affinity chromatography.C57BL/6 mice(12 mice in each group) were immunized with MOGIgd-TrxA,TrxA or saline.Clinical scores and histopathology were measured to evaluate the establishment of experimental autoimmune encephalomyelitis(EAE) model.RESULTS: The 32 kD MOGIgd-TrxA fusion protein was localized to inclusion bodies,and the purity was about 95% after purified by metal chelate affinity chromatography.The mice immunized with recombinant human MOG developed chronic non-remitting EAE,with 75%(9/12)positive rate.The time of illness onset in average was(12.14±2.04) days after immunization.Histological examination showed inflammatory lesions and demyelination in brain and spinal cord.CONCLUSION: Recombinant human MOG is produced in E.coli as a thioredoxin fusion protein.The fusion protein has high immunogenicity and successfully induces EAE in C57BL/6 mice.更多还原